The present invention relates generally to the field of particle analyzers, and more specifically to the display and analysis of data collected from particle analyzers.
Particle analyzers enable analysis of properties of particles, for example, individual cells, by subjecting them to an excitation light and measuring the resulting scattered and/or emitted light as detected by one or more light detectors. Different types of particle analyzers, such as flow cytometers and scanning cytometers, are described in the art. In a flow cytometer, for example, the excitation light beams may be stationary, while analyte cells in a liquid flow through a point at which the light beams converge. A scanning cytometer scans a fixed cell population, for example, on a microscope slide, with one or more excitation light beams. Flow cytometers can also be equipped with sorting devices that separate individual cells in a sample for further culture or analysis.
Prior to being exposed to an excitation light, particles may be labeled (also referred to as marked) with spectrally distinct fluorescent dyes or fluorescent dyes conjugated to molecule-specific ligands. In a sample, each particle may bind with one or more fluorescent dyes and/or dye conjugates. For example, a single cell may bind with one or more fluorescent dyes conjugated to antigen-specific antibodies depending on the characteristics of the proteins that are elements of that cell. When an excitation light is focused on a cell in a flow cytometer, the cell may scatter and/or emit light in several directions. The pattern of the scattered and/or emitted light allows one type of cell to be distinguished from another. The resulting fluorescence pattern is generally indicative of defined characteristics of the particles under analysis. Samples of particles are generally labeled with multiple dyes or dye conjugates in order to identify a range of properties of the constituent particles.
A particle analyzer may include multiple light detectors, and dyes are selected so that the peak fluorescence range of each dye is detected by a separate light detector. Due to the very low intensity of the kind of light emitted by small particles, these instruments are generally equipped with very sensitive detectors known as photomultiplier tubes (PMTs) that can detect individual photons. The resulting fluorescence measurements are based on the number of the photons detected by each light detector.
Often, when multiple dye fluorescence is being measured, the particle data contains some noise due to spillover of one fluorescence into another fluorescence range. The degree of spillover varies due to many factors, but in general, as more colors are being measured the noise due to spillover increases. Therefore, in many situations, the particle data is processed to first reduce the noise due to this spillover effect through a process known as compensation. Compensation, attempts to offset an estimated spillover amount from the particle data. Therefore, some particle data values resulting after compensation can be in the negative range.
Due to the large dynamic range of the measured fluorescence signal intensities, often in the range of four log decades or 10,000:1, linear display of a large amount of data would be of little clarity. In general, an exponential curve in the linear domain appears as a linear curve in the logarithmic domain. The logarithmic scale is therefore able to display a large dynamic range such as 10,000:1 and is used very frequently to display and analyze particle data. However, the data may contain a number of negative data points. Mathematically such negative data points are undefined on a logarithmic scale and are often lost or just shown as a collection of points on a display axis, limiting the usefulness of those data points.
Scaling systems, such as the biexponential model (Logicle) display described in U.S. Pat. No. 6,954,722, rely upon display techniques to present a useful display of the data addressing some issues of the pure logarithmic scale display mentioned above. The biexponential model applies a linear scaling for lower intensity data points and a logarithmic scaling for higher intensity data points that alleviates the problem of inability to show negative data points in a pure logarithmic scaled display.
However currently available methods generally require computation-intensive root finding operations and/or the use of lookup tables to determine the value to be used for display based on the particle data (including compensated particle data).
What is needed therefore, are methods and systems to flexibly and efficiently transform particle data in a manner that facilitates display and analysis of that data.